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II. CLONING TECHNIQUES

All of the cloning techniques are very similar and are based on the same pattern. The first one was called the nuclear transfer. It gave the bases of cloning but it has a very low success rate (to get a viable clone, about five hundreds experiments are necessary). The second one, called the Roslin technique, is quite the same as the nuclear transfer but it uses different donor cells. Finally, the last one, which is also the best one, the Honolulu

The nuclear transfer:

This technique requires two cells: first, an egg cell, unfertilized, and enucleated (the nucleus is taken out so there is no genetic material in the ovocyte). Then, we use a donor cell (in theory, every cell can be used). To simplify the process, the cell must not divide so it is forced into the G0 cell stage (also called the Gap Zero), which is a dormant phase.

Then, the nucleus of this donor cell is extracted and placed into the egg cell, which then produces an embryo. If it works, this embryo is transplanted into a surrogate mother (the female used to carry the embryo) and becomes a clone of the donor animal (because it has its genetic material).

The Roslin technique:

It is the way in which scientists cloned a sheep to create Dolly at the Roslin Institute, in 1997. This technique is not completely different from the nuclear transfer. They used an enucleated egg cell too but they took specific donor cells; as we have already said, every cell can be used for cloning but scientists noticed that udder cells worked better than others. Also, for this experimentation, the researchers allowed the cell to divide and so it formed an in vitro culture, so that the same nucleus was copied a hundred times (by mitosis). This step is useful for one reason (which has nothing to do with cloning) because scientists can alter the DNA to change the genetic material and the changes can be studied on the offspring (to know where the change has an effect and what it does). The donor cell is put into the G0 stage too (for this, the researchers starved the cell so that it could not divide). Then they used electric pulses to fuse the two cells together and activate the development of an embryo (with the previous technique, transferring the donor’s cell nucleus was quite a difficult process and the embryo did not always develop) <see picture 1>.

But the problem is that only a few electrically activated cells survive long enough to produce an embryo. Finally, the embryo is placed into the uterus of a surrogate mother <see picture 2> and the newborn animal is an exact copy of the donor.

The Honolulu Technique:

Created by the University of Hawaii in 1998, two years after the birth of Dolly, this technique enables mice to be cloned with a much higher success rate (3 clones out of 100 attempts) than with the previous technique (1 out of 277). They used specific donor cells which naturally remain in the G0 state (so they don’t have to starve the cell, which can easily destroy it) such as Sertoli cells, brain cells and cumulus cells (for example, mitosis for human brain cells lasts 80 years....). The nucleus of the donor cell is then placed into an unfertilized and enucleated egg cell. They wait for an hour (time for the egg cell to accept the nucleus) before placing in a chemical culture to jumpstart the cell’s growth, just as fertilization does in nature <see picture 3>

After being jumpstarted, the cell becomes an embryo and is transplanted into a surrogate mother. You know the end... This technique is the most powerful and viable one because it has a high success rate (it uses specific donor cell and a chemical culture to fuse the egg cell and its new nucleus and to help the cell to grow). Using this technique, the clones created were able to reproduce whereas this was not the case with the nuclear transfer and the Roslin Technique. They first cloned mice and this experimentation allowed new discoveries about genetics because the genome of mice is best known and they reproduce much more rapidly than sheep.

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